【作者】 刘凤沂；

【导师】 沈晋良；

【作者基本信息】 南京农业大学 ， 农药学， 2008， 博士

【摘要】 一、棉铃虫抗性品系的筛选及抗性等位基因频率的鉴定继续采用转Bt基因棉叶喂饲法对棉铃虫室内抗性品系YCR进行筛选,从第73代至88代,该品系对Bt毒素的抗性从2523倍上升到7311倍,继续筛选到104代,保持着极高水平抗性。采用等位基因特异性PCR技术（PASA）对室内抗性品系棉铃虫的钙粘素大片断缺失基因（r₁）进行鉴定,其基因型r₁r₁,且其频率为1。二、建立抗性棉铃虫的检测标准室内比较了棉铃虫抗、感亲本及正反交后代在转Bt基因棉叶上5天的发育速率。结果表明:抗、感亲本及正反交后代在转Bt基因棉叶上5天的存活率与发育速率存在明显差异,抗性品系的存活率达到75%,其中65%（存活幼虫的87%）存活幼虫龄期达2龄中期以上,体重≥0.6 mg/头,正反交和敏感品系的存活率仅为6%（活虫体重0.1-0.5mg/头,龄期为1-2龄初）、2%（活虫体重0.1-0.3 mg/头,龄期为1龄）及0%。因此,确定棉铃虫在转Bt基因棉叶上5天发育到2龄中期以上、体重≥0.6 mg/头的个体作为判别抗性纯合子的标准。三、棉铃虫在转Bt基因棉上发育速率的研究采用叶片喂饲法比较抗性品系和敏感品系在转Bt基因棉和常规棉上的生长发育,结果表明:在室内条件下,该高抗品系棉铃虫用转Bt基因棉和常规棉叶片喂饲5天的存活率差异不显著,均为80%以上;继续喂饲棉叶,抗性棉铃虫在转Bt基因棉叶上能够完成整个生长发育,但仅有6.7%的羽化率。在转Bt基因棉上幼虫历期较在常规棉上长6天,其化蛹率、蛹重、羽化率等均较常规棉低,表明抗性品系在转Bt基因棉上生长发育存在明显的生存劣势。由于抗性与敏感棉铃虫生长发育的速率不一致以及在一个长期的转基因作物的选择压力下产生高抗的棉铃虫能够在转Bt基因棉上存活,并有少量个体能完成整个生长发育,这将影响到庇护区内大量发育速率快的敏感个体与转Bt基因棉田少量发育速率慢的抗性个体的正常随机交配,从而可能削弱了庇护区治理策略的治理效果。四、棉铃虫对转Bt基因棉的抗性遗传及抗性连锁分析采用饲料感染法测定了21%MVPII可湿性粉剂（WP）和20%MVPII胶悬剂（SC）对棉铃虫抗（YCR）、感（YCS）亲本及其正反交（F₁,F′₁）后代的毒力,21%MVPIIWP对正反交后代的毒力分别为12.031（95%CI:7.972-18.263）μg/mL和6.617（95%CI:4.337-9.960）μg/mL;20%MVPII SC对正反交后代的毒力分别为61.659（95%CI:43.05-90.55）和98.22（95%CI:76.73-123.65）μg/mL。两毒素对正反交后代的毒力差异均不显著（95%置信限重叠）,表明其抗性为常染色体遗传。根据Stone公式计算得到21%MVPII WP对正反交后代的显性度分别为-0.51和-0.69（20%MVPII SC的显性度分别为-0.61和-0.44）,二者均在-1～0之间,证实具有7000多倍极高水平抗性的YCR品系对Cry1Ac的抗性基因（r₁）为不完全隐性遗传。正交后代雌虫与抗性亲本雄虫回交（BC₁）后代、正交后代雄虫与抗性亲本雌虫回交（BC₂）后代分别用转Bt基因棉叶处理5d后,其基因型分离比例接近1∶1,经分子检测得到进一步验证,证明棉铃虫对转Bt基因棉的钙粘素大片断缺失基因（r₁）也是常染色体遗传,且该钙粘素大片断缺失基因（r₁）与抗性紧密连锁。五、田间棉铃虫的抗性监测2005、2006、2007年从河北邱县和威县（2005年）转Bt基因棉大田灯诱采集第二代棉铃虫雄虫采用F₁代法在室内用转Bt基因棉植株或棉叶喂饲法检测田间棉铃虫对表达单价Cry1Ac毒素的转Bt基因棉的抗性等位基因频率。三年期间分别检测了来自邱县棉田86、127和135头雄虫,其中分别有8、24和29头田间亲本检测到携带抗性等位基因,估测抗性等位基因的频率分别为0.047（95%CI:0.015-0.079）、0.094（95%CI:0.044-0.145）和0.107（95%CI:0.055-0.159）;2005年采用F₁代法检测威县32头田间雄性成虫检测到1头携带有抗性等位基因,其抗性等位基因频率为0.016（0-0.0595）。2005年采用F₂代法从邱县131头田间雌性成虫中检测到7头携带有抗性等位基因,其频率值为0.01539（95%CI:0.0067-0.0277）,单雌系抗性等位基因检出概率值(1-P_NO)为93.8%。2005年两种方法检测邱县的结果基本一致,说明F₁和F₂代法均能检测到田间稀少的、携带隐性抗性杂合子的个体,与F₂代法相比,F₁代法是更为有效、经济、实用的田间抗性检测方法。在2007年又用剂量-反应法测定田间种群的抗性。结果表明:该种群对20%MVPII SC产生13.14倍抗性,为中等水平抗性。这些结果警示邱县棉铃虫种群的抗性已明显上升,长期大面积种植表达单价Cry1Ac毒素的转Bt基因棉和缺少非Bt棉做为有效庇护区可能加速了田间抗性进化,应在该地区尽快制定和实施有效的抗性治理策略。更多还原

【Abstract】 1.The selection of resistance strain YCR and detecting freqency of resistance alleleThe resistant strain（YCR） was selected with Bt cotton leaves from 73 to 88 generation,and resistance rate to Cry1Ac protoxin was increased from 2523-fold of 73 generation to 7311-fold of 88 gerenation.The YCR strain was subsequently selected to 104 generation in the laboratory and maintained the extremely high level of resistance to Bt. thuringiensis.In addition,using PASA（PCR amplification of specific alleles） method,we identified the deletion mutation gene of cadhern-like（r₁） in YCR and detected the frequency of the resistance allele,the result showed that the frequency of resistance allele （r₁）in YCR strain approached 1.2.Establishing of standard of detection resistant cotton bollwormComparing wtih the Bt-resistance and Bt-susceptible cotton bollworm on Bt cotton leaves for 5d,the result showed that Bt-susceptible and Bt-resistant H.armigera had significantly difference in larval surviving rate and growth rate on Bt cotton leaves. Resistant individuals（r₁r₁）had considerably higher survival rate（75%）,and a majority of survivors（65%of the total or 87%of the survivors）reached≥0.6mg/larvae and at least mid-2^nd instar.Whereas the surviving rate of reciprocal crosses of YCR and YCS strain,and YCS strain were 6%（0.1-0.5mg/larvae weight,from first instar to early of second instar）, 2%（0.1-0.3mg/larvae weight,first instar）and 0%,respectively.Therefore,individuals which reached＞0.6 mg/larvae and beyond mid-2^nd instar on Bt cotton leaves for 5 days, were considered as the homozygous resistant individual standard.3.The growth rate of H.armigera on Bt cotton leavesComparing with the development of Bt-resistant and Bt-susceptible bollworm（YCR and YCS）on Bt cotton leaves for 5 days,both the surviving rates of YCR on Bt cotton and non-Bt cotton leaves were 80%,which was not obviously different.6.7%survivors could developed adults by continuously feeding with Bt cotton leaves,and the development period of Bt-resistant larvae on Bt cotton leaves was delayed for 6 days than on non-Bt cotton.The pupation rate,pupa weight and emergence rate were lower than on non-Bt cotton,which indicated that the Bt-resistant insect suffered a survival disadvantage on Bt cotton leaves relative to its non-Bt counterpart.The asynchrony between susceptible and resistant populations and the surviving ability on Bt cotton under long-term selection pressure would influence the random mating between a lot of susceptible moths from refuge and few resistant moths from Bt cotton field,and weaken the efficiency of refuge strategy.4.Resistance inheritance and linkage analysis of resistance to Cry1Ac in H.armigeraUsing diet containing Bt toxin method,Logit regression analysis of Bt-resistant strain （YCR）,Bt-susceptible strain（YCS）and reciprocal crosses between them（F₁ and F’₁） of H. armigera indicated that LC₅₀ of the progenies from F₁ and F’₁ were 12.031（95%CI: 7.972-18.263）μg/mL and 6.617（95%CI:4.337-9.960）μg/mL for 21%MVPII WP; 61.659（95%CI:43.048-90.552）μg/mL and 98.217（95%CI:76.726-123.645）μg/mL for 20%MVPII SC,respectively,which there were no significantly difference between the two LC₅₀s.These results showed the gene that confers resistance to Bt toxin was on autosome. According to the formula of Stone（1968）,the dominance degree（D） of F₁ and F’₁ were -0.51and -0.69 for 21%MVPII WP（-0.61 and -0.44 for 20%MVPII SC）,respectively.The result indicated that the resistance to Cry1Ac toxin was inhetited as an incompeletely recessive trait（-1＞D＞0）.The genetype of progenies of two backcrosses BC₁(♀_F1×♂_YCR) and BC₂ and(♂_F1×♀_YCR) on Bt cotton leaves for 5d had 1:1 separation following the Mendel’s law,at same time,they were verified by using PASA method,results showed that the inheritance of resistance was on autosome too,and the truncated cadherin-like gene（r₁） in YCR strain was tightly linked to Cry1Ac resistance.5.Resistance monitoring of field populations of H.armigeraDuring 2005-2007,we estimated frequencies of alleles conferring resistance to Bt cotton by using the F₁ screen method in Qiuxian and Weixian（2005） counties.By feeding Bt cotton plants or leaves for 5 d,F₁ offspring from each single-pair line established in 2005-2007 were screened for resistance alleles based on larval growth,development,and surviving rate.8,24 and 29 collected-field male moths out of 86,127 and 135 lines were detected to carry resistance alleles in Qiuxian,and the frequencies of resistance alleles were estimated as 0.047（95%CI:0.015-0.079）、0.094（0.044-0.145）and 0.107（0.055-0.159）, respectively;1 collected-field male moth out of 32 lines were detected to carry resistance allele in Weixian,and the frequency of resistant allele Weixian was estimated as 0.016 （0-0.0595）.In 2005,7 collected-field female moths out of 131 iso-lines were detected to carry resistance alleles by using F₂ screen method in Qiuxian,and the frequency of resistance alleles was estimated as 0.01539（0.0067-0.0277）,the detection power of F₂ screen(1-P_NO) for lines screened was 93.8%.The results from the F₁ screen and F₂ screen methods in 2005 were consistant during detecting resistance of cotton bollworm population in Qiuxian,which indicated that the resistant herozygosis with rare and recessive gene can be detected by both F₁ screen and F₂ screen.Comparing with F₂ screen,the F₁ screen was more effective,applicative and low-cost method for monitoring resistance in field population.In 2007,the results from dose/response method showed that there was 13.14-fold resistance to 20%MVPII SC which means that there was a middle level resistance in this region.The results from several methods comfirmed that the resistance in field population had been increasing.Long-term use of Bt cotton expressing single Cry1Ac toxin and the lack of proper resistance management practice might accelerated the resistant evolution in field populations of the cotton bollworm.It is necessary to establish and implement effective resistance management strategy in this region as soon as possible.更多还原